Smad4 is a component of the Smad pathway that is involved in signal transduction in the TGF-β pathway (Levy, L and Hill, C. S., Molec. Cell. Biol. 25:8108-8125 (2005); Fukuchi, M. et al., Cancer 95:737-743 (2002)). This gene, also known as dpc4 (for “decreased in pancreatic carcinoma”), appears to be a tumor suppressor gene, and a decrease in smad4 expression has been observed in a variety of primary carcinomas, including pancreatic carcinomas (Luttges, J. et al., Am. J. Pathol. 158:1677-1683 (2001); Subramanian, G. et al., Cancer Res. 64:5200-5211 (2004)), esophageal carcinomas (Fukuchi, M. et al., Cancer 95:737-743 (2002), cervical carcinomas (Maliekal, T. T. et al., Oncogene 22:4889-4897 (2003), and other primary human cancers (Iacobuzio-Donahue, C. A. et al., Clin. Canc. Res. 10:1597-1604 (2004), as well as in cell line cancer models including of pancreatic cancers (Lohr, M. et al., Cancer Res. 61:550-555 (2001); Yasutome, M. et al., Clin. Exp. Metastasis 22:461-473 (2005)), and of colon cancers (Levy, L., and Hill, C. S., Molec. Cell. Biol. 25:8108-8125 (2005)). A reduced expression of smad4 in tumors has been associated with poor prognosis for patient survival, particularly in patients with smad4-deficient pancreatic adenocarcinomas (Liu, F., Clin. Cancer Res. 7:3853-3856 (2001); Tascilar, M. et al., Clin. Cancer Res. 7:4115-4121 (2001); Toga, T. et al., Anticancer Res. 24:1173-1178 (2004)). The mechanism of the tumor suppressive activity of the smad4 gene product is poorly understood, but it is thought that it may act as a “switch” regulating the growth-suppressive and growth-activating activities of certain components of the TGF-β signaling pathway (for reviews, see Akhurst, A. J., J. Clin. Invest. 109:1533-1536 (2002); Bachman, K. E., and Park, B. H., Curr. Opin. Oncol. 17:49-54 (2004); Bierie, B., and Moses, H. L., Nature Rev. Cancer 6:506-520 (2006)). Smad4 deficient tumors often respond less well to chemotherapy.
Therefore, there is a need to provide an alternative or improved therapy for treating SMAD4 deficient cancer.
Bone morphogenetic proteins (BMPs) are growth factors that belong the TGFβ superfamily. They comprise of around 20 members, classified into distinct subfamilies, depending on their sequence homology and functionality. BMP-2/BMP-4 and BMP-5/BMP-6/BMP-7 form subgroups that have been extensively studied and demonstrated to have osteogenic capacities (Wilson et al. 2013).
Although first identified by their capacity to support bone and cartilage development (Wozney et al. 1988), BMPs are involved in organ development and regulate homeostasis in a wide array of tissues. Besides their homeostatic function in normal embryonic and adult tissues, BMPs also play important roles in the pathology of several diseases, such as obesity, diabetes and cancer (Bragdon et al. 2011). They mediate their function by controlling differentiation, proliferation, cell growth and apoptosis at the intra and intercellular level. BMPs are secreted to the extracellular matrix as mature active dimers and can act on the target cells by binding to two molecules of type I (BMPR1a or BMPR1b) and two molecules of type II (BMPR2 or ActR2) Serine/Threonine kinase receptors, thereby forming an hexameric complex that will initiate the intracellular signaling cascade (Miyazono, Kamiya, and Morikawa 2010). Depending on the presence or absence of pre-formed BMPR complexes, the signaling will be mediated via the SMAD family following a defined canonical pathway or by less defined non-canonical intracellular effectors (Mueller and Nickel 2012). Translocation to the nucleus of these transcription factors, will initiate transcription of BMP-target genes.
The significance of the critical role of BMPs in many biological processes is perhaps best manifested by the presence of a multi-level regulation of BMP function. At the transcriptional level, control of BMP expression can be mediated by gene methylation (Kimura et al. 2008; Wen et al. 2006; Zhang et al. 2007), or by specific microRNAs, such as microRNA196b and mIR20b (Braig et al. 2010). At the cytosol, the presence of the smad inhibitors (iSMADs), that compete for receptor or SMAD binding such as Smad 6 and 7 can also attenuate BMP function. Other inhibitors such as protein phosphatases (PP1 and PP2) dilute the strength of BMP signal by dephosphorylating both the receptors and the pSMADs (Wrighton, Lin, and Feng 2009). BMP signaling can also be modulated by the presence of extracellular molecules that bind to BMP, inhibiting or enhancing BMP activity (Zakin and De Robertis 2010). Co-receptors potentiate BMP function by enhancing ligand binding whereas pseudoreceptors inhibit BMP signaling by sequestering BMPR2 and rendering it unavailable for BMP ligands (Onichtchouk et al. 1999). Another well studied group of extracellular BMP modulators is the Cysteine-knot group of BMP antagonists, which bind distinct BMPs with high affinities and prevent their interaction with the receptors. Depending on the structure (size of the cysteine knot) they are divided into three groups: the DAN family (Gremlin, Sclerostin), the twisted gastrulation (Tsg), and chordin and noggin.
The interplay between BMPs and their antagonists is crucial in determining their effects in development and in adult tissues. For several years the role of specific BMPs has been addressed in many nondiseased and diseased tissues. One of the most intriguing and poorly understood BMPs is BMP4. BMP4 seems to have opposing roles in cancers (Kallioniemi 2012). In certain malignancies high levels of BMP4 seems to be associated with less malignant features while in, for instance, breast and colon cancers BMP4 non canonical signaling was involved in EMT and metastatic behavior (Guo, Huang, and Gong 2012; Voorneveld et al. 2014). A more recent observation is the role of BMP4 on cell differentiation and epithelial metaplasia (Mari et al. 2014). To resolve the actual role of BMPs in different disorders it is required to study their specific function in diverse disease models.
The option of regulating BMP signaling at the extracellular receptor level is highly attractive, since extracellular regulation of signaling can be easily targeted to manipulate its function. Therefore, several natural antagonists have been tested to study the BMP effects on cellular processes. This approach is, however, hampered by the fact that the natural BMP inhibitors are limited to study the effect of the individual BMPs due to several factors intrinsic to their mechanism of action. In general, natural BMP antagonists are non selective in nature and modulate signaling of not only different BMPs, but also other members of the TGFB family, such as activins and nodal (Balemans and Van Hul 2002). Thus, studies using natural antagonists might not reflect results from the inactivation of individual BMPs. Moreover, the multi-target antagonism is not limited to the TGFB family members, as most antagonists can also interfere with wnt signaling pathways which indirectly affect BMP function (Yanagita 2005). Also several mechanisms of action have been described for some of these antagonists, rendering these molecules highly unspecific at inhibiting BMP function. For instance, Chordin possesses BMP-independent functions due to its binding to cell surface proteins and, thus, altering of cellular integrity (Chen et al. 2004). In some cases, BMP modulators possess such opposing modes of regulation, that they can act both as anti- or pro-BMP function, like in the case of Tsg and CV2 (Gazzerro and Canalis 2006; Oelgeschläger et al. 2000; Rentzsch et al. 2006; Wills, Harland, and Khokha 2006). Another complicating factor is that their activity is in turn tightly regulated by extracellular factors, including other BMP antagonists and other proteins, such as the metalloproteases Xolloid and Tolloid (Piccolo et al. 1997). Thus, if the expression of antagonists overlaps, it might result in activation of BMP signaling instead of inhibition. Therefore, these natural antagonists are ill-suited to inhibit or to define the individual action of each BMP and to study the role of specific BMPs in different disorders.
There are also antibodies available which bind to BMP4, anti-BMP4 R&D (Clone 66119, Mouse IgG2B, MAB757) and anti-BMP4 Abcam (Rabbit polyclonal, ab39973). In WO2008030611 antibodies are described which have an affinity for BMP2 and BMP4. Disadvantage of existing anti-BMP4 antibodies is that they have a low affinity for BMP4, are less capable of inhibiting BMP4 signaling and/or functionally blocking BMP4 in-vivo. It is an objective of the invention to overcome one or more of these disadvantages.